OBJECTIVES: Use of single dose nevirapine (SD NVP) for prevention of HIV-1 mother-to-child transmission (pMTCT) is associated with selection of K103N-containing HIV variants. Repeat use of SD NVP for pMTCT may influence emergence and persistence of NVP-resistant variants. DESIGN: K103N-containing variants were studied in 48 Ugandan women who received SD NVP in the HIVNET 012 trial, and were re-exposed to SD NVP in one (n = 44) or two (n = 4) subsequent pregnancies during a 5-year follow-up study. METHODS: Samples were analyzed using the LigAmp assay (assay cutoff: 0.5% K103N). RESULTS: Among 44 women who were re-exposed to SD NVP in one subsequent pregnancy, 37.8% had K103N detected within 1 year of SD-NVP re-exposure. Detection of K103N was independently associated with detection of K103N 6-8 weeks after the first SD NVP exposure and with pre-NVP viral load. The portion of women with undetectable K103N by 2 years after SD NVP administration was similar after first versus second use of SD NVP for pMTCT. K103N was undetectable in 93.2% of evaluable women by 3 years of re-exposure. Only two of four women who received SD NVP in two pregnancies during the follow-up study had K103N detected after the last SD NVP exposure. CONCLUSIONS: K103N was detected in some women within 1 year of SD NVP re-exposure, but faded from detection in most women by 3 years after re-exposure. Detection of K103N by 1 year after SD NVP re-exposure was associated with prior selection of K103N-containing variants and with pre-NVP viral load.